Generation of hepatocytes from pluripotent stem cells

ABSTRACT

Methods are provided for producing differentiated cells from stem cells, including producing hepatocytes. Compositions thereof are also provided, as are methods of treating a liver disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.14/409,234, filed Dec. 18, 2014, which is a U.S. national stage entryunder 35 U.S.C. 071 of PCT International Patent Application No.PCT/US2013/048113, filed Jun. 27, 2013, which claims benefit of U.S.Provisional Patent Application No. 61/666,219, filed Jun. 29, 2012, thecontents of each of which are incorporated herein by reference into thesubject application.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersDK071111, DK088561, DK041296 and CA013330 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inparentheses by number. Full citations for these references may be foundat the end of the specification. The disclosures of each of thesepublications, and of all patents, patent application publications andbooks cited herein, are hereby incorporated by reference in theirentirety into the subject application to more fully describe the art towhich the subject invention pertains.

The liver occupies a central position in life due to its crucialmetabolic, synthetic, storage, and drug or toxin disposal functions.Isolated liver cells are extremely useful for developing disease models,as well as for toxicological testing and drug development. Moreover,because many proteins are made in liver cells, cell/gene therapydirected at the liver is of extensive interest for a long list ofgenetic or acquired conditions. However, shortages of donor organs haveproved to be an insurmountable hurdle for therapeutic and otherapplications of liver cells. Therefore, alternative means to generatehepatocytes, e.g., from pluripotent stem cells, is of great interest.This requires understanding into the processes by which pluripotent stemcells may transition and differentiate, first into immature and theninto mature hepatocytes. Among candidate pluripotent stem cells ofinterest, human embryonic stem cells (hESC) or induced pluripotent stemcells (iPS), which share properties of the former, divide indefinitelyand may differentiate to produce mature cells of various tissues andorgans. However, available differentiation protocols to generatehepatocytes from hESC or iPS, etc., are inefficient and generate cellsof indeterminate developmental or maturational stages. For instance, theconvention of generating hepatocytes from aggregation of hESC or othertypes of pluripotent stem cells to form embryoid bodies is not onlyinefficient, but yields complex lineage mixtures at variousdevelopmental stages or maturity that pose difficulties in isolatingcells of interest, which may be additionally altered or damaged by cellseparation procedures (1). Directed differentiation of stem cells intohepatocytes could overcome these problems, (2, 3), but thisaccomplishment has generally been elusive.

The present invention addresses the need for directed differentiation ofstem cells into hepatocytes.

SUMMARY OF THE INVENTION

A method of producing a differentiated cell from a pluripotent stem cellis provided, the method comprising maintaining the pluripotent stem cellin a medium comprising conditioned medium from immortalized fetalhepatoblasts, for a time sufficient to produce the differentiated cell.

A composition is provided for differentiating stem cells into adifferentiated cell of interest, the composition comprising conditionedmedium obtained from a culture of hepatoblasts cultured in a mediumcomprising a basal medium.

A composition is provided for differentiating stem cells into adifferentiated cell of interest, the composition comprising componentsidentified in conditioned medium obtained from a culture of human fetalhepatoblasts cultured in a medium comprising a basal medium withoutthose components.

A method is provided for treating a liver disorder in a subjectcomprising administering to the subject an amount of the describedcompositions, or an amount of the described differentiated cells, in anamount effective to treat a liver disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1H. Morphology of hESC cultured with FH-CM. (A) UndifferentiatedhESC with clusters of small cells. (B) Primary embryonic/fetalhepatocyte-like cells (eFHLC) (P0) after 14d with fetalhepatocyte-conditioned medium (FH-CM). Note larger cell size andepithelial morphology. Inset, higher magnification showing binucleation,common to hepatocytes. (C-F) eFHLC with glycogen, G6P, glucagon andvimentin. (G-H) eFHLC subpassaged once (P1) or twice (P2). Orig. Mag.,×200.

FIG. 2A-2C. Characterization of differentiating eFHLC. (A) Light andelectron microscopy (LM, EM) in eFHLC and fetal human hepatocytesshowing similarities in epithelial properties (EM, bottom; magnificationbar=1 μm). (B) RT-PCR for gene expression in hESC, d14 eFHLC and freshlyisolated fetal human hepatocytes. Arrowheads indicate endodermal andmesodermal markers in eFHLC. (C) Temporal gene expression profile byqRT-PCR in eFHLC over d0, d3 and d14. Pluripotency markers, OCT4, NANOGand SOX2 declined, endodermal markers, SOX17, FOXA2, and mesodermalmarker, brachyrury, increased transiently, while hepatic transcriptionfactors, GATA4, HNF4, HNF1A, were increasingly expressed. Hepatic genes,i.e., AFP, ALB, AAT, TTR, were coordinately expressed, along withbiliary marker, CK19, and mesenchymal markers. Cluster analysis ofglobal gene expression in hESC, eFHLC, freshly isolated fetal humanhepatocytes (FH-PP) or cultured fetal human hepatocytes (FH-P3), andadult human hepatocytes (AH), showed convergence of eFHLC most towardsFH-P3.

FIG. 3A-3C. Microarray analysis of gene expression in eFHLC versus hESCand freshly isolated fetal human hepatocytes (FH-PP). Panels A, B showglobal differences in gene expression. Data were from total genesequences called as present, range, 48,942 to 51,031. In eFHLC, 505 or2123 genes were uniquely upregulated versus hESC and FH-PP,respectively, and 236 or 1514 genes were uniquely downregulated versushESC and FH-PP, respectively. By contrast, 1504 genes were uniquelyupregulated and 1387 genes were uniquely downregulated in FH-PP cellsversus hESC cells. This indicated that eFHLC were closer to hESCcompared with FH-PP. Table in 3C provides the overall distribution ofdifferentially expressed genes and fractions representing major geneontology groups and Kyoto Encyclopedia of Genes and Genomes (KEGG)pathways. Changes in TGF-β signaling and BMP signaling in eFHLC wereobserved compared with FH-PP cells. The data indicated TGF-β and BMPsignaling were more active in eFHLC. This was in agreement with similarfindings after FH-PP had been cultured.

FIG. 4A-4F. Secretory, synthetic, metabolic and hepatoprotectivefunctions in cultured eFHLC. (A-C) Studies with hESC, d14 eFHLC andHepG2 hepatoma cells for albumin secretion, urea synthesis and CYP450activity showing gain of these functions in eFHLC. (D) Assay of TNF-αcytotoxicity in primary mouse hepatocytes showing eFHLC-CM protectedcells. Asterisks indicate p<0.05 versus hESC (A-C), or untreatedcontrols (D). (E-F) Show regulation of ataxia telangiectasia mutated(ATM) signaling in cells. After 15 μM cis-P, a known inducer ofdouble-strand DNA breaks, viability of ATMP-tdT Huh-7 cells, declined(C) and ATM promoter activity increased (D), confirming DNAdamage-induced ATM signaling. When cells were treated with cis-P plusFGF1, FGF2, GCSF and IGF1, cell viability improved and ATM promoteractivity decreased. VEGF was ineffective. Asterisks, p<0.05, versuscis-P-treated cells.

FIG. 5A-5C. RayBiotech array analysis of 507 proteins in eFHLC-CM. (A)Basal medium not exposed to cells. (B) eFHLC-CM harvested after 24 h.Boxes marked by (+) and (−) in A and B indicate positive and negativecontrols. Each protein was represented twice in arrays. (C) Proteinsfound in eFHLC-CM are listed.

FIG. 6A-6C. RayBiotech array analysis of proteins in FH-CM. (A) Showsbasal medium containing various additives but without exposure to cells.(B) FH-CM harvested after 24 h. Boxes marked by (+) and (−) in A and Bindicate positive and negative array controls. Each protein wasrepresented twice in arrays. (C) Shows categorization of proteins inFH-CM according to density of array spots to indicate higher or lowerlevels.

FIG. 7A-7D. RayBiotech array analysis of receptor tyrosine kinase (RTK)expression in undifferentiated hESC. (A) Untreated control hESC withbasal phosphorylation of several receptor tyrosine kinases (RTKs). (Band C) hESC stimulated for 1 h (B) or 6 h (C) with FH-CM before analysisof phosphorylated RTKs. Data showed no differences from untreatedcontrols in (A). Each protein was spotted twice. Boxes marked by (+) and(−) in A-C indicate positive and negative array controls. (D) List ofphosphorylated RTKs in hESC irrespective of basal or FH-CM-stimulatedconditions.

FIG. 8A-8B. Differentiation of hESC over 3d with FH-CM altered byprotracted heating to degrade proteins or passage through Amiconmembrane of 3 Kd cut-off size. (A) hESC after culture with heatdenatured FH-CM showing switch to epithelial morphology. (B) hESCcultured with FH-CM passed through Amicon membrane showing epithelialmorphology. Orig. Mag., ×100.

FIG. 9A-9F. Hepatic differentiation of hESC with group of 7 CP. Panelson left show undifferentiated hESC, panels in middle show hESC culturedwith FH-CM and panels on right show hESC cultured with combination of 7CP, which were most effective for this purpose. (A) Phase contrastmicroscopy showing undifferentiated hESC with small size of cells(left), whereas hESC cultured with FH-CM (middle) or 10 μm amounts of 7CP showed larger size and epithelial morphology (on right). (B-D)Immunofluorescence staining of hESC under conditions indicated for OCT4,albumin and vimentin. This showed loss of OCT4 and gain of albumin andvimentin expression after differentiation (red color). Nuclei werestained blue with DAPI. (E & F) eFHLC derived from hESC by either FH-CMor CP showed urea synthesis (E) as well as cytochrome P450 activity withconversion of ethoxyresorufin to resorufin (E). Human HepG2 cells wereincluded for comparisons in E & F.

FIG. 10A-10F. Hepatic differentiation of iPSC with group of 7 CP. Panelson left show undifferentiated iPSC, panels in middle show iPSC culturedwith FH-CM and panels on right show iPSC cultured with combination of 7CP, which were most effective. (A) Phase contrast microscopy showingundifferentiated iPSC with small size of cells (left), whereas iPSCcultured with FH-CM (middle) or 10 μm amounts of 7 CP showed larger sizeand epithelial morphology (on right). (B-D) Immunofluorescence stainingof iPSC under conditions indicated for OCT4, albumin and vimentin. Thisshowed loss of OCT4 and gain of albumin and vimentin expression afterdifferentiation (red color). Nuclei were stained blue with DAPI. (E & F)eFHLC derived from iPSC by either FH-CM or CP showed urea synthesis (E)as well as cytochrome P450 activity with conversion of ethoxyresorufinto resorufin (E). Human HepG2 cells were included for comparisons in E &F.

DETAILED DESCRIPTION OF THE INVENTION

A method of producing a differentiated cell from a pluripotent stem cellis provided, the method comprising maintaining the pluripotent stem cellin a medium comprising isolated conditioned medium from hepatoblasts,for a time sufficient to produce the differentiated cell.

A method of producing a differentiated cell from a pluripotent stem cellis provided, the method comprising maintaining the pluripotent stem cellin a medium comprising isolated conditioned medium from fetalhepatoblasts, for a time sufficient to produce the differentiated cell.

Also provided is a method of producing a differentiated cell from apluripotent stem cell, the method comprising maintaining the pluripotentstem cell in a medium comprising conditioned medium from hepatoblasts,or a medium comprising two or more of phenacetin, phytosphingosine HCl,and pyridoxal HCl, for a time sufficient to produce the differentiatedcell. In a preferred embodiment, the method comprises maintaining thepluripotent stem cell in a medium comprising two or more of phenacetin,phytosphingosine HCl, and pyridoxal HCl.

In an embodiment of the methods, the differentiated cell is ahepatocyte. In an embodiment, the differentiated cell exhibits ameso-endodermal phenotype of a fetal human hepatocyte. In an embodiment,the differentiated cell exhibits ureagenesis and/or albumin synthesisand/or vimentin expression. In an embodiment, the pluripotent stem cellis an inducible pluripotent stem cell or is an embryonic stem cell.

In an embodiment of the methods, the hepatoblasts are immortalized. Inan embodiment, the hepatoblasts have been immortalized by contact with atelomerase. In an embodiment, the hepatoblasts have been immortalized byexpression of telomerase, or maintenance of telomerase expression. In anembodiment, hepatoblasts are human. In an embodiment, hepatoblasts arefetal. In an embodiment, hepatoblasts are immortalized human fetalhepatoblasts. In an embodiment, hepatoblasts are isolated. In anembodiment, the medium does not comprise serum. In an embodiment, theconditioned medium from immortalized human fetal hepatoblasts comprisesmedium obtained from a culture of immortalized human fetal hepatoblastscultured in a medium comprising a basal medium. In an embodiment, theimmortalized fetal hepatoblasts are human immortalized fetalhepatoblasts. In an embodiment, the hepatoblasts are mammalian, but arenot human. In an embodiment, the hepatoblasts are human, but are notfetal. In an embodiment, the medium comprising a basal medium furthercomprises L-glutamine, one or more non-essential amino acids, and anantibiotic. In an embodiment, the basal medium is a Dulbecco's ModifiedEagle's Medium (DMEM). In an embodiment, the antibiotic ispenicillin-streptomycin. In an embodiment, the non-essential amino acidsare glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid,L-proline and L-serine. In an embodiment, the medium comprising a basalmedium further comprises an artificial serum replacement.

A conditioned medium, with regard a culture, is a medium in which thecell culture has been maintained. In an embodiment, the conditionedmedium has been exposed to the cells being cultured for 1 or more hours,2 or more hours, 6 or more hours, 12 or more hours, 24 or more hours,one week or more or two weeks or more.

In an embodiment of the methods, the medium further comprises one ormore of L-cysteinglutathione disulfide, γ-Glu-Cys, DL-kynurenine,D-penicillamine disulfide, and tetracaine HCl. In an embodiment, themedium does not comprise tetracaine HCl. In an embodiment, the mediumcomprises L-cysteinglutathione disulfide, γ-Glu-Cys, DL-kynurenine,D-penicillamine disulfide, phenacetin, phytosphingosine HCl, andpyridoxal HCl.

In an embodiment of the methods, the medium comprises retinoic acidand/or dexamethasone. In an embodiment, the medium comprisesL-glutamine. In an embodiment, the medium comprises a selenium compound.In an embodiment, the medium comprises sodium selenite. In anembodiment, the medium comprising a selenium compound also comprises oneor more of an albumin, transferrin, insulin, progesterone, putrescine,biotin, 1-carnitine, corticosterone, ethanolamine, d(+)-galactose,glutathione (reduced), linolenic acid, linoleic acid, retinyl acetate,selenium, T3 (triodo-1-thyronine), dl-α-tocopherol, dl-α-tocopherolacetate, catalase, and superoxide dismutase. In an embodiment, proteinsand enzymes of the medium are isolated from human or are recombinantwith a human sequence.

A composition is provided for differentiating stem cells into adifferentiated cell of interest, the composition comprising isolatedconditioned medium obtained from a culture of hepatoblasts cultured in amedium comprising a basal medium. Basal media are widely-known in theart, and as used herein are understood to encompass cell-growth media(for example, un-supplemented) used for culturing mammalian cells.

A composition is provided comprising isolated conditioned mediumobtained from a culture of hepatoblasts cultured in a medium comprisinga basal medium, or a basal medium further comprising two or more ofphenacetin, phytosphingosine HCl, and pyridoxal HCl.

A composition is provided for differentiating stem cells into adifferentiated cell of interest, the composition comprising conditionedmedium obtained from a culture of hepatoblasts cultured in a mediumcomprising a basal medium, or a basal medium further comprising two ormore of phenacetin, phytosphingosine HCl, and pyridoxal HCl. In anembodiment, (i) the medium comprising a basal medium from which theconditioned medium is obtained, or (ii) the basal medium furthercomprising two or more of phenacetin, phytosphingosine HCl, andpyridoxal HCl, also further comprises L-glutamine, non essential aminoacids, and an antibiotic.

In an embodiment of the compositions, the medium comprising a basalmedium further comprises L-glutamine, non essential amino acids, and anantibiotic. In an embodiment, the basal medium is a DMEM. In anembodiment, the antibiotic is penicillin-streptomycin. In an embodiment,the non-essential amino acids are glycine, L-alanine, L-asparagine,L-aspartic acid, L-glutamic acid, L-proline and L-serine. In anembodiment, the medium comprising a basal medium further comprises anartificial serum replacement.

In an embodiment of the compositions, the medium further comprises oneor more of L-cysteinglutathione disulfide, γ-Glu-Cys, DL-kynurenine,D-penicillamine disulfide, and tetracaine HCl. In an embodiment, themedium does not comprise tetracaine HCl. In an embodiment, the mediumcomprises L-cysteinglutathione disulfide, γ-Glu-Cys, DL-kynurenine,D-penicillamine disulfide, phenacetin, phytosphingosine HCl, andpyridoxal HCl. In an embodiment, the aforelisted components are,independently, present at a concentration of 10 μM or less, of 7.5 μM orless, 5 μM or less, of 2.5 μM or less, or 1 μM or less. In anembodiment, the aforelisted components are, independently, present at aconcentration of greater than 0.01 μM.

In an embodiment of the compositions, the medium comprises retinoic acidand/or dexamethasone. In an embodiment, the medium comprisesL-glutamine. In an embodiment, the medium comprises a selenium compound.In an embodiment, the medium comprises sodium selenite. In anembodiment, the medium comprising a selenium compound also comprises oneor more of an albumin, transferrin, insulin, progesterone, putrescine,biotin, 1-carnitine, corticosterone, ethanolamine, d(+)-galactose,glutathione (reduced), linolenic acid, linoleic acid, retinyl acetate,selenium, T3 (triodo-1-thyronine), dl-α-tocopherol, dl-α-tocopherolacetate, catalase, and superoxide dismutase. In an embodiment, proteinsand enzymes of the medium are isolated from human or are recombinantwith a human sequence.

In an embodiment of the compositions, the hepatoblasts are immortalized.In an embodiment, the hepatoblasts are human hepatoblasts. In anembodiment, the hepatoblasts are fetal hepatoblasts. In an embodiment,the hepatoblasts are immortalized human fetal hepatoblasts. In anembodiment, the hepatoblasts are mammalian, but are not human. In anembodiment, the hepatoblasts are human, but are not fetal.

In the various media described herein and in the various compositionscomprising media, the specified components are present in amountsconsistent with the health and/or growth of the cells in the medium.

A hepatocyte is an epithelial parenchymatous cell of the liver. In anembodiment, the hepatocyte is capable of secreting bile. In anembodiment, the hepatocyte is polygonal. Induced pluripotent stem cellsare adult cells, typically somatic cells, that have been geneticallyreprogrammed to an embryonic stem cell-like state by being caused toexpress genes and factors important for maintaining the definingproperties of embryonic stem cells. In an embodiment, the inducedpluripotent stem cells are mammalian. In an embodiment, the adult cellfrom which the induced pluripotent stem cell is induced is a human adultcell. Embryonic stem cells are pluripotent stem cells derived from theinner cell mass of the blastocyst. In an embodiment, the embryonic stemcells are mammalian. In a further embodiment, the embryonic stem cellsare non-human. In an embodiment, the embryonic stem cells are human.Basal media are serum-free media widely available in the art.

A method is provided for treating a liver disorder in a subjectcomprising administering to the subject an amount of the describedcompositions, or an amount of the described differentiated cellsobtained by any of the methods described hereinabove, in an amounteffective to treat a liver disorder.

A liver disorder is a disorder or pathology of the mammalian liver whichimpairs the proper functioning of the liver as compared to a healthyliver. Such disorders are widely known in the art.

“And/or” as used herein, for example, with option A and/or option B,encompasses the separate embodiments of (i) option A, (ii) option B, and(iii) option A plus option B.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

Here, the principle is established that soluble signals from fetal livercells undergoing hepatic development and with stem or progenitor cellproperties can drive differentiation in pluripotent stem cells togenerate hepatocytes in the meso-endodermal stage of fetal liverdevelopment. These pluripotent stem cell-derived hepatocytes expressedrepertoires of hepatobiliary and mesenchymal genes, distinct microRNAprofiles, as well as synthetic and metabolic hepatic functions, e.g.,albumin secretion, urea production, and xenobiotic disposal, whichrecapitulated properties of fetal hepatocytes. Therefore, thesehESC-derived cells were designated “embryonic/fetal hepatocyte-likecells” (eFHLC). It was found that hepatic functions were expressed atsufficient levels in eFHLC to sustain mice with fatal drug-induced acuteliver failure (ALF) (11). In this setting, transplanted eFHLC providedliver support to prolong life. Moreover, eFHLC released paracrinefactors that were capable of protecting hepatocytes in vitro and thismechanism promoted liver regeneration in vivo to complete rescue of micewith drug-induced ALF. Therefore, this method to manipulate pluripotentstem cells to generate hepatocytes in defined developmental stage andwith appropriate hepatic potency offers opportunities for a variety ofcell type-specific basic, clinical and other applications.

Experimental Results Example 1

Differentiation of pluripotent hESC generated hepatocytes: First, it wasdetermined in what ways would soluble signals from immature fetalhepatocytes (FH) direct differentiation of hESC. To avoid donor-to-donorvariability, FH immortalized by telomerase were used, withstem/progenitor cell properties, as well as meso-endodermal phenotype ofprimary FH (12-13). Conditioned medium (CM) was harvested from FH over24 h and excluded serum. Feeder cells or animal additives were notincluded. A multi-step protocol was developed that included culture ofhESC in FH-CM alone for first 3d followed over the next 3d by culture ofhESC with FH-CM plus three known soluble factors and then furtherculture of hESC for a total of 14d with FH-CM plus another known solublefactor. None of the soluble factors added to FH-CM was essential forinitiating hepatic differentiation in hESC. However, in the presence ofthese known soluble factors, hESC adhered better in plastic culturedishes.

During differentiation, hESC in primary culture (P0) gained epithelialmorphology within 3d (FIG. 1A-1B). The eFHLC expressed liver/pancreasforegut endoderm markers, i.e., glycogen, glucose-6-phosphatase (G6P),glucagon, and others, and the mesenchymal marker, vimentin (VIM) (FIG.1C-F). Over 14d, 0.50±0.07×10⁶ hESC originated 1.94±0.05×10⁶ eFHLC,which constituted a 4-fold gain in cell numbers. Moreover, populationdoublings of eFHLC during P1 and P2 cultures (FIG. 1G-1H),produced >20-fold gains in their cell numbers, which indicated theability of differentiating cells to continue proliferating, and wasconsistent with highly efficient generation of differentiated cells frompluripotent stem cells.

eFHLC were characterized by morphology, gene expression and functionalassays. eFHLC acquired more cytoplasm, larger nuclei, even binucleatedcells, similar to hepatocytes (FIG. 2A). Cytoplasmic complexity withlysosomes, microperoxisomes and vacuoles, resembled that in hepatocytes.Reverse transcription-polymerase chain reactions (RT-PCR) showed hepaticmarkers, i.e., α-fetoprotein (AFP), albumin (ALB), G6P, α-1-antitrypsin(AAT), cytokeratin (CK)-18, metabolic enzymes, e.g., CYP-1B1, -3A4,-2E1, and -1A1, and also mesenchymal markers, i.e., VIM, α-smooth muscleactin (aSMA) (FIG. 2B), confirming conjoint meso-endodermal phenotype ofnatural fetal hepatocytes (6,7). Temporal differentiation profile byquantitative RT-PCR was informative, as within 3d,pluripotency-associated genes, i.e., OCT4, NANOG and SOX2, wereexpressed less, and endoderm-associated genes, i.e., SOX17, FOXA2, andearly mesoderm-associated gene, brachyury, were expressed more (FIG. 2C,Table 2). By 14d of epithelial advancement, E-cadherin (ECAD), a markerof epithelial cells, was well-expressed, which was paralleled by theappearance after 3d and increase after 14d of hepatic transcriptionfactors, GATA4, HNF1a and HNF4a. Hepatic gene expression changedcorrespondingly, since AFP appeared after 3d, and ALB, AAT or TTRincreased between 3d and 14d. Similarly, expression of CYP3A4 andCYP7A1, which characterize mature hepatocytes, increased during thisperiod. Nonetheless, VIM and CK19 expression after 14d assigned eFHLC tothe fetal hepatic stage. Global gene expression profiling by Affymetrixmicroarrays in hESC, eFHLC, primary or cultured fetal hepatocytes, andadult hepatocytes, substantiated this possibility, since eFHLC divergedfrom undifferentiated hESC and converged more along fetal than adulthepatocytes (FIG. 2D). In eFHLC, gene expression ontology and regulationof cytokine signaling trended toward fetal hepatocytes (FIG. 3). GlobalmicroRNA (miRNA) profiling showed several similarities between eFHLC andfetal hepatocytes when array analysis of cellular miRNA in eFHLC versushESC and freshly isolated fetal human hepatocytes (FH-PP) was performed.

TABLE 2 qRT-PCR analysis of gene expression - Gene expression wasnormalized with β-actin and represents fold-change versusundifferentiated hESC on d 0 of differentiation protocol: Undiffer-entiated eFHLC Gene analyzed H1-hESC d 0 3 d 14 d Pluripotency markersOCT4 1.0 0.1 0 NANOG 1.0 0.1 0 SOX2 1.0 0.1 0 Endodermal markersBRACHYURY 1.0 10 0.6 SOX17 1.0 56 1.0 FOXA2 1.0 27 0.2 Epithelial markerCDH1 1.0 1.5 2.6 Mesenchymal marker VIM 1.0 1 11 Hepatic transcriptionalfactors GATA4 1.0 155 488 HNF4A 1.0 165 653 HNF1A 1.0 58 136 Hepaticmarkers AFP 1.0 2 0.4 ALB 1.0 7 21 AAT 1.0 18 82 TAT 1.0 20 25 TTR 1.037 273 TDO2 1.0 20 29 ASGPR1 1.0 1 1.6 APOF 1.0 7 9 CYP3A4 1.0 67 66CYP7A1 1.0 12 14 G6P 1.0 40 68 Biliary marker CK19 1.0 8 17

Protein studies verified mRNA findings in eFHLC as stem cell markersdeclined (OCT4, NANOG, TRA-1-80) and hepatic (FOXA2, ALB, G6P andglycogen) and biliary (GGT) properties increased. In d14 eFHLC, OCT4 waslost with gain of ECAD, VIM, FOXA2, and ALB expression. eFHLC containedhepatobiliary markers, glycogen, G6P and GGT. Similarly, epithelial(ECAD) and mesenchymal (VIM) markers were coexpressed. High levelexpression of asialoglycoprotein receptor (ASGPR1), which is specific toadult hepatocytes, indicated many eFHLC were maturing along the hepaticlineage. Flow cytometry showed 27% eFHLC expressed ASGPR1, markingmature hepatocytes.

eFHLC expressed hepatic synthetic, metabolic and xenobiotic disposalfunctions in vitro: After 14d of differentiation, eFHLC secretedalbumin, synthesized urea and converted a xenobiotic, ethoxyresorufin,to resorufin (FIG. 4A-4C). Such functions are important for hepaticsupport in liver failure (7, 11). Similarly, paracrine factors mayprotect hepatocytes from injury (14). This was confirmed since CM fromeFHLC protected mouse hepatocytes from tumor necrosis factor (TNF)-αtoxicity (FIG. 4D). Recently, it was established that drug-induced ALFin mice involved impairment in ataxia telangiectasia mutated (ATM)signaling (11), which is regulated by paracrine signals. Therefore, todemonstrate whether soluble factors could restore ATM signaling,lentivirally-modified Huh-7 cells were prepared (from humanhepatocellular carcinoma) to express the tdTomato reporter gene undercontrol of cloned human ATM promoter (15). When these hATMP-tdT cellswere cultured with cis-platinum (Cis-P), DNA strand breaks were induced,cell viability declined and ATM promoter activity increased (FIG. 4E,4F). It was found that culture of hATMP-tdT cells with Cis-P, plus FGF1,FGF2, G-CSF and IGF1 but not VEGF, improved cell viability and loweredATM promoter activity. These factors had been identified to be presentin eFHLC-CM (FIG. 5), which indicated that eFHLC were capable ofaffecting intracellular signaling through paracrine mechanisms.

eFHLC rescued mice with drug-induced ALF: Identification of hepaticfunctions and potential for paracrine signaling suggested eFHLC couldsupport recovery of the damaged liver. This was facilitated by aNOD/SCID mouse model of ALF (11), where rifampicin (Rif), phenytoin(Phen) and monocrotaline (MCT) caused dysregulation of Atm signaling,leading to severe oxidative stress, DNA damage, hepatic necrosis, livertest abnormalities, coagulopathy, encephalopathy, and 90-100% mortality.Intraperitoneal transplantation of mature hepatocytes with microcarrierscaffolds rescued mice with ALF. Of note, transplanted hepatocytesremained in peritoneal cavity without migrating to the liver. Moreover,reseeding of the liver with cells was unnecessary. ALF was induced withRif-Phen-MCT in mice (n=20), followed by 4-6×10⁶ eFHLC (n=10) or vehicle(n=10) i.p. In the FHLC group, 5 mice survived (50%) versus only 1 mousein the vehicle group (10%), p<0.001. In eFHLC-treated mice,encephalopathy was absent or less severe, whereas sham-treated micedeveloped severe encephalopathy (grade 3-4), p<0.05. Liver testsimproved in the eFHLC group versus the vehicle group. After 7d, serumalanine aminotransferase (ALT) was 77±82 versus 4800±500 u/l and totalbilirubin 0.5±0.2 versus 2.5±0.5 mg/dl, p<0.05. Normal blood glucose andserum creatinine levels in all mice excluded hypoglycemia or renalfailure as coincidental causes of death. Human cells were identified incell-microcarrier conglomerates recovered from peritoneal cavity.Histological sections of microcarrier (mc) and cell-conglomeratesrecovered from mice 7d after eFHLC transplantation showed vascularreorganization (H&E staining), glycogen in transplanted cells, andconfirmation of human transplanted cells by in situ hybridization forprimate-specific centromere sequences. Transplanted eFHLC were absentfrom the native liver, as was expected. Livers of vehicle-treated micewere edematous, hemorrhagic and necrotic with extensive expression ofphosphorylated histone H2AX, confirming oxidative DNA damage, and onlyinterspersed Ki-67+ cells, indicating limited liver regeneration. IneFHLC-treated mice, liver necrosis and H2AX expression decreased, whilethe prevalence of Ki-67+ cells increased. Histological grading showed5.4-fold less liver injury in eFHLC-treated mice after 7d, 0.7±0.3versus 3.8±0.4, p<0.05. The prevalence of Ki-67+ cells at that time was2.3-fold greater in eFHLC-treated mice, 91±5 versus 39±4 cells/HPF,p<0.05. Analysis of hepatic gene expression indicated extensiveperturbations in animals with ALF (Table 3). In eFHLC-treated mice,expression of genes in oxidative/metabolic stress, inflammatorycytokines, chemokines or other mediators, and of Atm and cell cycleregulators, i.e., Ccnc, Ccnd1 or Egr1, improved.

After i.p. or subcutaneous transplantation, eFHLC did not proliferate,and no tumors were observed in NOD/SCID mice over 3 months.Undifferentiated hESC generated teratomas as expected (not shown) (5-7).

TABLE 3 Liver mRNA expression by qRT-PCR (fold NOD/SCID mice versus micein ALF with sham-treatment or of eFHLC transplantation - Data were firstnormalized against housekeeping β-actin gene in individual samples. Eachcondition was analyzed with samples in triplicate Gene Sham- Sham-eFHLC- eFHLC- Gene description Symbol 3 d 7 d 3 d 7 d Oxidative orMetabolic Stress Crystallite, alpha B Cryab −1.2 1.6 1.1 −1.1 CytochromeP450, family 1, subfamily a, polypeptide 1 Cyp1a1 −3.7 −4.9 −5.8 −4.7Cytochrome P450, family 1, subfamily b, polypeptide 1 Cyp1b1 −3.4 1.5−2.6 −1.3 Cytochrome P450, family 2, subfamily a, polypeptide 5 Cyp2a5−6.7 −5.1 −1.1 −1.9 Cytochrome P450, family 2, subfamily b, polypeptide10 Cyp2b10 −1.2 1.1 −1.7 −1.8 Cytochrome P450, family 2, subfamily b,polypeptide 9 Cyp2b9 −7.3 −7.2 −7.3 −2.1 Cytochrome P450, family 2,subfamily c, polypeptide 29 Cyp2c29 −46.3 −106.9 −1.4 −1.4 CytochromeP450, family 3, subfamily a, polypeptide 11 Cyp3a11 −13.8 −27.7 −9.1−4.8 Cytochrome P450, family 4, subfamily a, polypeptide 10 Cyp4a10 1.6−5.9 −13.9 −11.0 Cytochrome P450, family 4, subfamily a, polypeptide 14Cyp4a14 7.8 −2.0 −7.8 −18.8 Cytochrome P450, family 7, subfamily a,polypeptide 1 Cyp7a1 −32.9 −100.2 −64.1 −11.7 Epoxide hydrolase 2,cytoplasmic Ephx2 −4.1 −10.4 −2.6 −2.0 Flavin containing monooxygenase 1Fmo1 −15.6 −2.3 −1.9 −1.3 Flavin containing monooxygenase 4 Fmo4 −1.2−2.0 −2.1 −2.2 Flavin containing monooxygenase 5 Fmo5 1.7 −1.3 1.0 −1.6Glutathione peroxidase 1 Gpx1 −3.2 −3.1 −3.0 −2.1 Glutathione peroxidase2 Gpx2 −1.6 −1.1 −1.7 1.0 Glutathione reductase Gsr −1.4 −1.4 −2.1 −2.0Glutathione S-transferase, mu 1 Gstm1 −3.8 −10.6 −5.8 −2.5 GlutathioneS-transferase, mu 3 Gstm3 −3.9 −5.8 −1.1 1.8 Heme oxygenase (decycling)1 Hmox1 1.1 3.5 2.2 −1.1 Heme oxygenase (decycling) 2 Hmox2 −1.2 −1.6−2.4 −3.0 Metallothionein 2 Mt2 5.7 7.8 17.8 −2.1 Polymerase (RNA) II(DNA directed) polypeptide K Polr2k −1.5 −1.2 −2.7 −2.3 P450(cytochrome) oxidoreductase Por −1.7 −5.9 −2.5 −4.4 Superoxide dismutase1, soluble Sod1 −2.7 −3.6 −7.5 −6.0 Superoxide dismutase 2,mitochondrial Sod2 −4.4 −5.4 1.3 −1.1 Heat Shock DnaJ (Hsp40) homolog,subfamily A, member 1 Dnaja1 −1.9 −2.8 −3.9 −3.4 Heat shock factor 1Hsf1 −1.4 −1.7 −1.8 −1.7 Heat shock protein 1B Hspa1b −1.9 −1.1 −1.5−2.0 Heat shock protein 1-like Hspa1l −2.8 −4.0 −7.4 −3.7 Heat shockprotein 4 Hspa4 −1.5 −3.3 −1.7 1.0 Heat shock protein 5 Hspa5 1.9 1.5−1.1 −1.4 Heat shock protein 8 Hspa8 −1.0 −1.2 −1.4 −1.4 Heat shockprotein 1 Hspb1 −1.2 1.8 −1.7 −2.0 Heat shock protein 1 (chaperonin)Hspd1 −1.6 −2.4 −1.7 −1.7 Heat shock protein 1 (chaperonin 10) Hspe1−1.7 −1.7 −2.4 −2.3 Proliferation and Carcinogenesis Colony stimulatingfactor 2 (granulocyte-macrophage) Csf2 −1.2 2.6 −1.4 1.1 Cyclin C Ccnc−1.7 −1.7 3.6 4.2 Cyclin D1 Ccnd1 −1.1 1.8 6.7 12.9 Cyclin G1 Ccng1 1.42.4 1.2 1.9 E2F transcription factor 1 E2f1 −1.0 2.2 1.4 2.2 Earlygrowth response 1 Egr1 4.2 15.3 30.6 9.7 Proliferating cell nuclearantigen Pcna −1.3 1.4 −1.0 −1.0 Growth Arrest and SenescenceCyclin-dependent kinase inhibitor 1A (P21) Cdkn1a 105.0 92.0 141.4 116.5DNA-damage inducible transcript 3 Ddit3 1.2 3.9 2.1 1.1 Growth arrestand DNA-damage-inducible 45 alpha Gadd45a −3.1 1.6 −2.1 −5.7Insulin-like growth factor binding protein 6 Igfbg6 −10.2 −3.5 −101.8−110.4 Transformed mouse 3T3 cell double minute 2 Mdm2 −1.0 1.8 5.4 4.7Transformation related protein 53 Trp53 −1.3 1.3 −1.1 −1.1 InflammationChemokine (C-C motif) ligand 21b Ccl21b −5.0 −16.6 −16.1 −16.6 Chemokine(C-C motif) ligand 3 Ccl3 5.4 30.7 6.3 4.4 Chemokine (C-C motif) ligand4 Ccl4 3.4 21.5 8.1 3.7 Chemokine (C-X-C motif) ligand 10 Cxcl10 2.410.8 4.5 1.1 Interleukin 18 Il18 −3.0 −2.1 −3.6 −2.9 Interleukin 1 alphaIl1a −2.9 1.0 −1.3 −1.2 Interleukin 1 beta Il1b 1.2 3.5 1.6 1.4Interleukin 6 Il6 1.9 7.3 6.0 1.8 Lymphotoxin A Lta −1.2 1.1 — −1.8Macrophage migration inhibitory factor Mif −1.5 −1.3 1.9 1.3 Nuclearfactor of kappa light polypeptide gene enhancer Nfkb1 −1.3 1.5 −1.3 −1.5in B-cells 1, p105 Nitric oxide synthase 2, inducible Nos2 6.5 24.4 26.51.8 Serine (or cysteine) peptidase inhibitor, clade E, member 1 Serpine150.8 57.3 286.0 7.4 DNA Damage and Repair Ataxia telangiectasia mutatedhomolog (human) Atm −1.7 −2.4 −1.3 1.1 CHK2 checkpoint homolog (S.pombe) Chek2 −1.1 1.6 −1.2 −1.1 Excision repair cross-complementingrodent repair deficiency, Ercc1 1.1 2.1 1.2 −1.1 complementation group 1Excision repair cross-complementing rodent repair deficiency, Ercc4 −1.5−2.0 −2.4 −2.1 complementation group 4 RAD23a homolog (S. cerevisiae)Rad23a −2.0 −2.7 −3.0 −2.8 RAD50 homolog (S. cerevisiae) Rad50 −1.8 −1.51.3 1.3 UDP glucuronosyltransferase 1 family, polypeptide A2 Ugt1a2 −2.8−3.1 −3.2 −2.5 Uracil DNA glycosylase Ung −1.7 −1.4 1.9 −1.4 X-rayrepair complementing defective repair in Chinese Xrcc1 −1.3 −1.4 −2.8−2.4 hamster cells 1 X-ray repair complementing defective repair inChinese Xrcc2 1.4 1.1 1.7 1.4 hamster cells 2 X-ray repair complementingdefective repair in Chinese Xrcc4 −1.1 −1.0 −1.4 −1.6 hamster cells 4Apoptosis Signaling Annexin A5 Anxa5 2.9 5.0 4.4 2.4 Bcl2-associated Xprotein Bax 1.5 2.3 1.2 1.2 Bcl2-like 1 Bcl2l1 1.7 2.6 3.9 1.9 Caspase 1Casp1 −2.4 1.7 −1.5 −1.2 Caspase 8 Casp8 −4.3 −1.1 −1.1 1.1 Fas ligand(TNF superfamily, member 6) Fasl −1.2 1.2 −1.7 −1.8 Nuclear factor ofkappa light polypeptide gene enhancer Nfkbia −1.3 1.7 −1.1 −1.4 inB-cells inhibitor, alpha Tumor necrosis factor receptor superfamily,member 1a Tnfrsf1a 1.1 1.5 1.7 1.1 Tumor necrosis factor (ligand)superfamily, member 10 Tnfsf10 −4.3 −3.4 −6.3 −4.9 TNFRSF1A-associatedvia death domain Tradd −1.6 −1.3 −2.3 −1.9

Mechanisms in hepatic differentiation of hESC: To understand how FH-CMcaused hepatic differentiation in hESC, cytokines and growth factorswere examined, and 62 of 507 such proteins were found to be present inFH-CM (FIG. 6), including regulators of cell differentiation, e.g.,activinA, FGFs, or transforming growth factors (TGF) (12, 13). As signaltransduction through these molecules should have engaged receptors oncell surface followed by phosphorylation of receptor tyrosine kinases(RTKs), 71 RTKs were examined in hESC stimulated with FH-CM for 10 min,1 h or 6 h. Surprisingly, RTKs were not activated, including receptorsof 12 ligands present in FH-CM (FIG. 7). To determine whether proteinsin FH-CM were dispensable for hESC differentiation, FH-CM was degradedby heating to 100° C., and passed FH-CM through Amicon membranes toremove >3 kilodalton size proteins. Protein-depleted FH-CM still inducedepithelial differentiation in hESC, including changes in morphology andgene expression, including in expression of pluripotency (Nanog, Sox-2),epithelial (AFP), or mesenchymal (VIM) genes within 3d (FIG. 8). It wasconcluded that nonprotein molecules in FH-CM were involved. Thesemolecules were stable since FH-CM generated eFHLC despite storage ofFH-CM for 6 weeks at 4° C. To characterize the nature of nonproteincomponents in FH-CM, the Purdue University Metabolite Profiling Facilityperformed LC-MS metabolomics analysis, which identified 810 compoundswith good separations in blank medium and FH-CM. Of these 810 compounds,105 compounds were >3-fold abundant in FH-CM versus blank medium, p<0.03(Table 4).

Genes expressed under differentiation conditions were analyzed byqRT-PCR with customized array from SA Biosciences. Expression ofindividual genes was normalized against housekeeping gene, β-actin. Thedata indicated culture of hESC with protein-depleted FH-CM was effectivein initiating differentiation along meso-endodermal hepatic stage. Mostnotable were decreases in expression of Nanog and Sox2 and increases inexpression of AFP and VIM.

TABLE 1 Changes in gene expression. (C) Genes expressed underdifferentiation conditions. Gene expression was analyzed by qRT-PCR withcustomized array from SA Biosciences. Undiffer- FH-CM-treated eFHLC x 3d entiated Heat- 3 kd Amicon Gene analyzed H1-hESC denatured cut-offPluripotency genes OCT4 1.0 1.3 1.3 NANOG 1.0 0.3 0.1 SOX2 1.0 0.7 0.3Endoderm markers BRACHYURY 1.0 0.5 1.9 SOX17 1.0 0.7 1.0 FOXA2 1.0 0.31.2 Epithelial marker CDH1 1.0 0.7 0.9 Mesenchymal marker VIM 1.0 6.24.6 Hepatic transcription factors GATA4 1.0 0.5 1.5 HNF4A 1.0 1.0 2.0HNF1A 1.0 0.4 1.4 Hepatic markers AFP 1.0 1.3 2.5 ALB 1.0 0.7 2.0 AAT1.0 0.5 1.8 TAT 1.0 0.6 1.9 TTR 1.0 0.5 1.7 TDO2 1.0 0.6 1.7 ASGPR1 1.00.9 1.5 APOF 1.0 0.5 1.9 CYP3A4 1.0 0.6 1.4 CYP7A1 1.0 0.7 3.3 G6P 1.00.5 2.0 Biliary marker CK19 1.0 0.7 0.6

TABLE 4 Listing of compounds identified by LC-MS metabolomics analysisin FH-CM Compound, Retention Corrected Compound Exact Mass Time (min)p-value Identification 281.1072 1.93 1.37E−09 281.1072 448.0098 2.001.22E−09 448.0098 426.0855 2.02 2.94E−10 Cysteineglutathione disulfide197.0828 2.15 3.49E−12 N-benzylideneaniline n-oxide 374.1351 2.176.77E−13 374.1351 380.1687 2.25 5.35E−09 Asn Thr Phe 211.0454 2.251.13E−09 211.0454 156.0031 2.50 2.26E−11 C6 H7 N P S 128.0112 2.511.08E−13 128.0112 268.0192 2.53 3.57E−13 268.0192 161.0683 2.54 1.55E−052-Aminoadipic acid 226.9919 2.64 1.92E−10 226.9919 1092.4396 2.651.06E−10 1092.4396  339.0923 2.66 1.17E−12 Cys Ser Met 380.1361 2.677.25E−04 C15 H29 N2 O3 P S2 408.1783 2.67 2.01E−05 Tryptophan 373.15322.71 2.22E−12 C15 H20 N9 O P 135.0547 2.71 1.44E−14 Adenine 301.11362.84 7.53E−10 C9 H15 N7 O5 210.9971 2.85 3.14E−09 210.9971 380.1367 2.901.01E−10 C15 H29 N2 O3 P S2 + 2.9005 90.0319 2.96 2.24E−16 Lactic acid323.0060 3.00 4.68E−13 C24 H4 P 167.0584 3.08 7.45E−14 Pyridoxal(Vitamin B6) 250.0623 3.47 3.36E−11 gamma-L-Glutamyl-L- cysteine122.0487 3.90 1.71E−09 Niacinamide 201.1666 4.04 2.92E−12 C12 H25 S149.0510 4.05 3.76E−10 Methionine 268.0713 4.72 9.69E−05 Homolanthionine444.1428 4.75 1.39E−08 444.1428 396.0489 5.00 4.58E−12 C21 H8 N4 O5329.9910 5.07 3.49E−12 C19 H9 P3 214.0085 5.09 4.95E−12 2,3-Dioxogulonicacid 296.0869 5.35 9.46E−15 Penicillamine disulfide 233.1224 6.303.27E−09 C11 H23 O P2 401.1190 9.24 1.77E−07 C17 H25 N2 O5 S2 612.153511.45 4.66E−10 Glutathione, oxidized 208.0851 12.83 8.30E−13 Kynurenine159.0685 15.31 2.39E−15 Indoleacetaldehyde 266.0351 32.20 1.27E−08 C13H15 P S2 482.1143 34.69 6.19E−13 C16 H26 N4 O9 S2 383.1070 35.602.47E−15 Succinoadenosine 135.0558 35.62 1.08E−13 C7 H7 N2 O 297.089835.63 4.09E−15 5′-Methylthioadenosine 313.0839 35.63 2.22E−13 C13 H12 N7O P 369.4807 36.70 2.74E−10 369.4807 315.2040 36.76 4.82E−12 C15 H23 N8499.1462 37.07 9.72E−11 C23 H28 N5 O2 P3 249.5732 37.30 7.64E−10249.5732 595.1771 37.42 2.15E−11 8-Hydroxy-perphenazine glucuronide1482.8310 37.65 1.69E−09 1482.831  556.1732 37.71 3.76E−12 C23 H28 N9 O4P2 639.1682 38.11 3.28E−13 639.1682 179.0950 38.20 2.24E−16 Phenacetine264.1823 38.21 3.28E−12 Tetracaine 312.1465 38.38 3.54E−13 Phe Phe514.1626 38.43 2.11E−10 C21 H26 N9 O3 P2 252.0902 38.49 5.52E−11Carbamazepine 10,11- epoxide 485.0762 38.74 5.00E−06 C27 H19 N O4 S21471.5754 38.79 3.76E−10 1471.5754  450.8249 38.79 6.24E−11 450.82491024.2646 38.80 5.22E−10 1024.2646  374.1240 38.81 1.14E−11 C27 H19 P771.3008 38.83 7.29E−12 771.3008 916.2433 38.89 2.75E−09 FMNH 346.099238.95 7.64E−10 C25 H14 O2 582.1468 38.99 3.49E−12 C27 H25 N10 P3590.1166 38.99 6.94E−12 5-Aminoimidazole ribonucleotide 574.1458 39.101.39E−08 C28 H26 N6 O4 S2 450.8238 39.32 1.38E−10 450.8238 578.206839.32 3.49E−12 C36 H37 O P3 265.0948 39.45 6.24E−11 265.0948 262.077639.54 1.57E−06 C8 H16 N5 O S2 574.1232 39.67 4.57E−13 C24 H34 N O7 P2 S2558.1804 39.74 6.47E−07 C35 H30 N2 O S2 276.0570 39.83 3.89E−13 C15 H9N4 P 421.1670 39.83 1.87E−06 C13 H27 N9 O3 S2 208.5870 39.92 4.39E−09208.587  574.1265 40.06 3.42E−11 C28 H31 O7 P S2 558.1820 40.24 1.31E−06C23 H34 N4 O8 S2 447.1469 40.38 1.58E−10 C23 H30 N O2 P S2 622.191440.39 2.50E−10 622.1914 314.1083 40.70 3.28E−13 C21 H17 N P 558.181640.84 1.35E−07 C23 H34 N4 O8 S2 + 40.837997 290.1090 40.97 3.66E−11 C15H18 N2 O2 S 350.0953 41.14 6.53E−10 N-acetylaspartate 330.1974 41.319.31E− 14 C16 H30 N2 O3 S 507.1460 41.32 2.60E−11 C32 H29 P2 S 290.108641.42 2.29E−11 C15 H18 N2 O2 S + 41.415 706.1547 41.52 6.64E−11 706.1547324.1161 41.76 2.29E−10 acetohexamide 1170.3890 41.81 4.73E−12 1170.389 507.1476 42.27 2.81E−10 C30 H24 N2 O4 P 324.1154 42.45 4.93E−12 C19 H19N O2 P 317.2929 42.65 9.15E−12 Phytosphingosine 507.1482 43.29 2.20E−121-deoxy-1-[methyl[3-phenyl- 3-[4-(trifluoro- methyl)phenoxy]pro-pyl]amino]-b-D- Glucopyranuronic acid 382.1235 43.56 4.58E−12 C20 H22 N3O P2 366.1256 43.63 6.18E−05 C17 H22 N2 O5 S 906.3010 44.60 3.26E−10906.301  583.0971 45.71 4.27E−09 583.0971 354.2546 46.00 3.63E−125beta-Chola-3,8(14),11-trien- 24-oic Acid 541.1893 46.15 6.05E−08 C29H33 O8 S 234.1606 46.18 2.09E−10 3-n-decyl acrylic acid 517.3172 47.164.12E−11 Linolenoyl lysolecithin 541.3209 47.22 4.31E−10 541.3209Discussion

Direct differentiation of hESC with FH-CM rapidly and efficientlygenerated hepatocytes, leading to uniformity of hepatic development,including expression of hepatic transcription factors,coordinately-regulated hepatobiliary genes, epithelial markers, as wellas mesenchymal markers. This recapitulated the meso-endodermal stage ofnatural fetal hepatocytes (4-7). The extent of synthetic, metabolic andxenobiotic disposal functions further confirmed this developmental stagein eFHLC. Despite this immaturity, transplanted eFHLC rescued mice inALF by providing critical life-support and paracrine signals to aidliver repair/regeneration, which was similar to the capability in thissetting of mature hepatocytes (7, 11).

Previously, lack of knowledge or uncertainties in the hepatic lineagestage achieved made it difficult to interpret the efficacy of stem celldifferentiation protocols (1-3, 18-21). Use of FH-CM withoutanimal-derived materials, feeder cells or genetic manipulation toexpress multiple transcription factors avoided previous majorrestrictions (22). These attributes should be especially helpful fordeveloping further insights into liver cell differentiation mechanismsin stem cells, as well as various applications of stem cell-derivedliver cells.

FH-CM induced hepatic differentiation in hESC by the steps of endodermspecification followed by maturation to fetal stage. This reproducedparacrine effects of proteins during differentiation of mouse stem cellsin vitro (17). However, hepatic differentiation induced by FH-CM lackingproteins was singularly different from cell differentiation induced bycytokine/chemokine/growth factor-based protocols. Despite the presencein FH-CM of proteins affecting cell attachment and perhapsproliferation, e.g., activinA, FGFs, GCSF, interleukin-6, TNF, VEGF,etc., (14,18,23,24), deficiency of RTK activation in hESC substantiatedthat these proteins did not play seminal roles in stem celldifferentiation with FH-CM. The findings indicated active role in stemcell differentiation of natural small compounds in FH-CM. Whether thesecompounds could additionally have emanated from eFHLC themselves duringcell differentiation was unknown. Previously, screening of chemicallibraries identified synthetic candidate compounds advancing β isletcell differentiation (25). These results are different from the presentfindings because small molecules in FH-CM originated naturally fromcells. Among the list of these small compounds, putativedifferentiation-inducing molecules included those affectingdifferentiation in stem/progenitor cells, e.g., 2-aminoadipic acid (26).Use of specific matrices and synthetic surfaces could help in furtherexpanding eFHLC or other stem cell-derived cell types.

Protein secretion, urea synthesis and xenobiotic metabolism in eFHLCindicated suitable hepatic properties. Cells engrafted and functioned inxenotolerant mice, which was similar to mature human hepatocytes (27).After transplantation, stem cell-derived hepatocytes may engraft andexpress liver functions in animals, although often largely at mRNA level(5-7, 22, 28) On the other hand, for cell therapy requiring organrepopulation with healthy cells, proliferation of transplanted cells iscritical (22, 28). In treating genetic conditions permanently,extensively modified cells, e.g., those reprogrammed with multipletranscription factors, will be less desirable than cells differentiatedsimply by extracellular soluble signals. It should be noteworthy that insettings, such as ALF, where short-term liver support by extrahepaticreservoirs of cells rescued animals without need for reseeding of theliver with cells, other considerations are applicable (7, 11). Forinstance, eFHLC showed capacity for glucose homeostasis, proteinsynthesis and ammonia detoxification, besides releasing hepatoprotectivesubstances, e.g., FGFs, GCSF, IGFs, VEGF, etc. Previously, only GCSF wasknown to regulate ATM promoter activity (29). It was found that FGFs andIGF also regulated hepatic ATM signaling. As molecular perturbations,including ATM signaling, improved after eFHLC transplantation, thisadded specificity to the effects of cell therapy in ALF.

Materials and Methods

Studies were approved according to NIH guidelines, by the EinsteinCommittee on Clinical Investigations, Embryonic Stem Cell ResearchOversight Committee, and Animal Care and Use Committee.

Fetal cells: Fetal livers were from Human Fetal Tissue Repository atEinstein. Ep-CAM+ liver cells were isolated and cultured as previouslydescribed (5, 6). hTERT-FH-B cells were cultured in DMEM with 10% FBS(12). To obtain CM, hTERT-FH-B were cultured for 24 h in DMEM/F12 mediumwith 2% Knock-out Serum Replacer (KSR), 2 mM L-glutamine, 0.1 mM MEM NonEssential Amino Acids (NEAA), 1% penicillin-streptomycin (InvitrogenCorp., Carlsbad, Calif.).

Culture of hESC. WA-01 hESC were passaged weekly on matrigel-coateddishes in DMEM/F12 medium, 1% B27 supplement, 1% N2 supplement, 2 mML-glutamine, 0.1 mM NEAA, 1% penicillin-streptomycin (Invitrogen Corp.)and 50 ng/ml basic FGF (R&D Systems, Minneapolis, Minn.). For the finalhepatic differentiation protocol, hESC were washed with DMEM/F12, andcultured in FH-CM.

Cytotoxicity assays. For effects of CM from hFHLC on TNF-α-inducedcytotoxicity, 1.5×10⁵ primary mouse hepatocytes were isolated bycollagenase perfusion (11), and plated in 24-well dishes in RPMI 1640medium with 10% FBS and antibiotics. After overnight culture, cells wereswitched to CM plus 10 ng/ml TNF-α (Sigma) for 16-18 h, followed bythiazolyl blue viability assays, as described previously (14). For cisPtoxicity assays, Huh-7 cells were used after transduction by alentiviral vector to express human ATM promoter-driven tdTomato gene(15). Huh-7 cells cultured with 15 μM cis-P (Sigma) or 2 μg/ml G-CSF(Amgen Inc., Thousand Oaks, Calif.) for 16-18 h were incubated for 20min with 6 μg/ml Hoechst33342 (Sigma) for cell viability andTdTomato/ATM promoter expression.

Hepatic functions. For albumin, cell culture medium harvested after 3 hwas analyzed by human albumin immunoassay (Bethyl Laboratories,Montgomery, Tex.). For ureagenesis, cells were incubated with 2.5-7.5 mMammonium chloride for 12 h and urea content was analyzed as previouslydescribed (27). For CYP450 activity, cells were induced overnight withphenobarbital, 7-ethoxyresorufin and μM dicumarol were added for 12 h at37° C., and resorufin was measured, as described previously (30).

Human cytokine arrays. Conditioned medium was analyzed by human antibodyarray I membrane for 507 human proteins and cell lysates were analyzedby Human RTK Phosphorylation Antibody Array 1 (RayBiotech, Norcross,Ga.), according to manufacturer.

NOD/SCID mice with ALF. CB17.NOD/SCID^(prkdc) mice, 6-7 weeks old, werefrom Jackson Labs. (Bar Harbor, Me.). Mice were given 3 daily doses ofi.p. Rif (75 mg/kg) and Phen (30 mg/kg) followed by one i.p. dose on d4of MCT (160 mg/kg), as described previously. ¹¹ After 1d, 4-6×10⁶hESC-derived cells differentiated for 14d were transplanted i.p. with 1ml Cytodex 3™ microcarriers (Amersham Biosciences Corp., Piscataway,N.J.). Sham-treated animals received microcarriers. Encephalopathy wasgraded from 0 (absent) to 3 (coma). Mice were observed for 2 weeks. Insome studies, hESC-derived cells were injected subcutaneously or i.p.for tumor formation over 3 months.

Immunohistochemistry. Cells were fixed in 4% paraformaldehyde inphosphate buffered saline, pH 7.4 (PBS), blocked/permeabilized with 5%goat serum, 0.2% Triton X-100 (Sigma) in PBS for 1 h, and incubatedovernight at 4° C. with anti-human antibodies for OCT3/4 (1:200), AFP(1:100), ECAD (1:50) (Santa Cruz Biotechnology Inc., Santa Cruz,Calif.), FOXA2 (1:100) (R&D Systems), ALB (1:200) (Sigma), VIM (1:100)(US Biologicals, Swampscott, Mass.). After washing in PBS,TRITC-conjugated goat anti-mouse IgG (1:50, Sigma) or anti-rabbit IgG(1:100) were added for 1 h with 4′-6-diamidino-2-phenylindole (DAPI)(Invitrogen) counterstaining. In negative controls, primary antibodieswere omitted. Glycogen, G-6-P, and GGT were stained as described (4-7).

Electron microscopy. Cells were fixed in 2.5% glutaraldehyde incacodylate butter, postfixed in osmium tetroxide, and stained with 1%uranyl acetate before embedding in plastic. Ultrathin sections wereexamined under JEOL 1200 electron microscope.

Molecular studies. RNA was extracted by TRIzol reagent (Invitrogen),cleaned by RNeasy (Qiagen Sciences, Germantown, Md.), incubated in DNaseI (Invitrogen) and reverse-transcribed by Omniscript RT kit (Qiagen).Platinum PCR SuperMix (Invitrogen) was used for PCR with annealing at94° C.×5 min, and 35 cycles at 94° C.×30 s, 55° C.×30 s, 72° C.×45 s,and 72° C.×10 min (primers, Table 4). Mouse Stress and Toxicity RT²Profiler PCR Array and RT² Real-Time SyBR Green PCR mix and RT² FirstStrand kit were from SABiosciences (Frederick, Md.). cDNA synthesis andPCR was according to the manufacturer. For quantitative (q) RT-PCRanalysis of gene expression, customized arrays were obtained for 24genes, including pluripotency genes, transcription factors andhepatobiliary genes (CAPH-0800A; SA Biosciences). Data were analyzed by2-ΔΔCt method. Fold-changes in gene expression were expressed aslog-normalized ratios from sham-treated/normal and celltransplantation/normal livers. Gene expression was analyzed with U1332.0 Plus oligonucleotide arrays (Affymetrix Corp., Santa Clara, Calif.)as described (10). Changes in mRNA expression were examined forpathway-specificity by IPA tools (Ingenuity Systems Inc., Redwood,Calif.).

TABLE 5 Primer sequences for RT-PCR (SEQ ID NOS. 1-24,top to bottom, respectively). Amplicon size Gene Primer sequence 5′-3′expected OCT4 F: GACAACAATGAAAATCTTCAGGAGA 218 bpR: TTCTGGCGCCGGTTACAGAACCA ALB F: TGCTTGAATGTGCTGATGACAGGG 161 bpR: AAGGCAAGTCAGCAGGCATCTCATC AFP F: TGCAGCCAAAGTGAAGAGGGAAGA 260 bpR: CATAGCGAGCAGCCCAAAGAAGAA CK-19 F: ATGGCCGAGCAGAACCGGAA 308 bpR: CCATGAGCCGCTGGTACTCC VIM F: CACCTACAGCCTCTACG 170 bpR: AGCGGTCATTCAGCTC α-SMA F: AGTACCCGATAGAACATGG 153 bpR: TTTTCTCCCGGTTGGC CYP1B1 F: CACCAAGGCTGAGACAGTGA 230 bpR: GCCAGGTAAACTCCAAGCAC CYP2C9 F: GGACAGAGACGACAAGCACA 200 bpR: TGGTGGGGAGAAGGTCAAT CYP3A4 F: TGTGCCTGAGAACACCAGAG 201 bpR: GCAGAGGAGCCAAATCTACC CYP2E1 F: CCGCAAGCATTTTGACTACA 202 bpR: GCTCCTTCACCCTTTCAGAC CYP1A1 F: AGGCTTTTACATCCCCAAGG 197 bpR: GCAATGGTCTCACCGATACA β-Actin F: TCACCACCACGGCCGAGCG 350 bpR: TCTCCTTCTGCATCCTGTCG Abbreviations: F, Forward; R, Reverse; bp, basepair; ALB, albumin; AFP, α-fetoprotein; CK-19, cytokeratin-19; VIM,vimentin; SMA, smooth muscle actin, CYP, cytochrome P450

For microRNAs, total RNA isolated by TRIzol reagent (Invitrogen) wasanalyzed by LC Sciences (Houston, Tex.) with probe set based on SangermiRBase, v 9.0. Later, transcripts with <500 arbitrary signals wereexcluded as these are not identified by qRT-PCR. Data were transformedto log 2 followed by clustering of transcripts (Cluster3, StanfordUniversity) and heatmaps were drawn (JavaTree1.1.6r2).

Tissue studies: Tissue samples were frozen to −80° C. in methylbutane.Cryostat sections were prepared. Tissue morphology was analyzed by H&Estained sections. Tissue injury was graded as previously described (38).For hepatic function in transplanted cells, glycogen and G-6-P werestained (35). For Ki67 and histone H2AX, tissues were fixed in 4% PAFfollowed by rabbit anti-Ki67 (1:750, Vector Laboratories, Burlingame,Calif.) or rabbit anti-phosphoS139 H2AX (1:300, ab2893; Abcam,Cambridge, Mass.), respectively, and secondary anti-Rabbit Alexa Fluor546 (1:500, Molecular Probes), followed by counterstaining with DAPI (7,11). Transplanted cells were identified by in situ hybridization foralphoid satellite sequences in centromeres (27).

Liquid chromatography-mass spectroscopy (LC-MS) metabolomics. Fornon-targeted metabolite screen by LC-MS, 5 uL media were injected andseparated on Agilent 1100 system with Waters Atlantis T3 column (3 μm,150×2.1 mm i.d.). Binary mobile phase consisting of solvent systems A(0.1% formic acid in ddH₂O, v/v) and B (0.1% formic acid inacetonitrile, v/v) were used in gradient elution with flow of 0.3mL/min. Initial conditions were set at 100:0 A:B, 1 min hold wasemployed followed by linear gradient to 70:30 at 20 min and lineargradient to 10:90 at 45 min. Gradient conditions were reset to 100:0 A:Bfrom 45.1 min to 55 min. After separation, column effluent wasintroduced by positive mode electrospray ionization into Agilent MSD-TOFmass spectrometer. Mass data from m/z 70-1100 were collected. Biomarkerswere identified with Agilent Mass Profiler Professional software, asrecommended. These studies were performed by Bruce Cooper at PurdueUniversity Metabolite Profiling Facility.

Serological studies. Sera were stored at −20° C. and analyzed for ALTand bilirubin as previously described. Human albumin was measured byimmunoassay (Bethyl Laboratories).

Statistical analysis. Data were analyzed by t-tests, Pearson correlationtests, logrank tests, and ANOVA with Holm-Sidak posthoc test. P values<0.05 were considered significant.

Example 2

Differentiation of human embryonic stem cells (hESC) by syntheticcounterparts of substances made naturally in fetal hepatocytes: The listof principal components identified by LC-MS in conditioned medium fromfetal hepatocytes (FH-CM), which was provided in Table 4 was furtherreduced to a set of 8 compounds (CP) (commercially available inchemically synthesized form) (Table 6).

TABLE 6 CP tested for induction of hepatic differentiation in hESC andiPSC CP Exemplary Catalog designation Chemical identity Manufacturer No.1 L-Cysteinglutathione Santa Cruz sc- Disulfide 211701 2 γ-Glu-Cys SigmaG0903 3 DL-Kynurenine Sigma 61250 4 D-Penicillamine disulfide SigmaP1101 5 Phenacetin Sigma 77440 6 Phytosphingosine HCl Sigma P2795 7Pyridoxal HCl Sigma P6155 8 Tetracaine HCl Sigma T7508

The ability of these CP to substitute for FH-CM in differentiating hESC(WA-01 line) was examined in various combinations of CP (see Table 7) inmicromolar concentrations of 1, 5, 10, 15, 20, and 25. Amounts of >10micromolar of the compounds caused toxicity with cell death anddetachment of hESC (WA-01 line) from surfaces of cell culture dishes.When these 8 CP were tested in hESC individually in medium (seemethods), none of the CP in 1, 5 or 10 micromolar amounts inducedhepatic differentiation, and OCT4 was still expressed, albumin orvimentin was not expressed, and cell morphology was unchanged. These 8CP were then tested in concentrations of 5 or 10 micromolar each incombinations to substitute for FH-CM in differentiating hESC.

TABLE 7 Combinations of CP and their effects on induction of hepaticdifferentiation in hESC and iPSC¶ Combination of compounds in cultureHepatic (+, present; CP CP CP CP CP CP CP CP differentiation −, absent)1 2 3 4 5 6 7 8 induced? 1 + − − − − − − − No (only undifferentiatedhESC) 2 + + − − − − − − No (only undifferentiated hESC) 3 + + + − − − −− No (only undifferentiated hESC) 4 + + + + − − − − No (onlyundifferentiated hESC) 5 + + + + + − − − Yes but incomplete(undifferentiated hESC present) 6 + + + + + + − − Yes but incomplete(undifferentiated hESC present) 7 + + + + + + + − Yes (most optimal withno obvious undifferentiated hESC) 8 + + + + + + + + Yes (but lessoptimal versus 7CP group of row above) ¶Hepatic differentiation wasevaluated by characteristic morphology of undifferentiated stem cellsand of epithelial cells along with albumin staining.

The culture medium contained additives, including retinoic acid,dexamethasone. Amounts of >10 micromolar of the compounds causedtoxicity with cell death and detachment of adherent hESC from surfacesof cell culture dishes. Further analysis indicated culture of hESC withCP individually or in groupings of up to 6 CP at one time in 1, 5 or 10micromolar amounts, did not alter morphology of cultured hESC and thesecontinued to display characteristic small sizes and cluster formation(not shown). By contrast, culture of hESC with a group of 7 CP in 5 or10 micromolar concentrations for 2 weeks generated large cells withuniform morphology of epithelial cells (FIG. 9A). The followingcombinations of CP were ineffective in hESC differentiation: CP1+CP2;CP1+CP2+CP3; CP1+CP2+CP3+CP4. The following combinations of CP werepartially effective because both undifferentiated hESC anddifferentiated cells were present in culture dishes: CP5+CP6; CP6+CP7;and CP5+CP6+CP7. By contrast, when 7 or 8 CP were combined together,only differentiated cells were present in culture dishes. Of these twocombinations, the following group of CP was most effective:CP1+CP2+CP3+CP4+CP5+CP6+CP7.

Further studies were performed of hESC-derived epithelial cells withthis set of 7 CP. In response to the most effective combination of 7 CP,morphology of undifferentiated hESC changed and expression of OCT4 wasno longer observed in differentiated hESC (FIG. 9A, 9B). Differentiatedcells showed albumin in cytoplasm indicating hepatic differentiation,whereas undifferentiated hESC were negative for albumin staining (FIG.9C). Moreover, differentiated cells expressed vimentin (FIG. 9D). Thedifferentiated cells generated by 7 CP synthesized urea (FIG. 9E) andpossessed ability to convert a xenobiotic, ethoxyresorufin, intoresorufin (FIG. 9F). These properties were similar to hESC-derived cellsgenerated by FH-CM (see Example 1).

iPSC were utilized that were obtained by reprogramming of normal humanfibroblasts with non-integrating Sendai virus vectors from thePluripotent Stem Cell Core at Albert Einstein College of Medicine. Thedifferentiation protocol with FH-CM and CP was identical to studies withhESC described above. After 2 weeks, iPSC cultured with FH-CM becamelarger with epithelial morphology, whereas undifferentiated iPSC weresmaller and were arranged in clusters (FIG. 10A). Similarly, iPSCcultured with above-described combination of 7 CP became larger withepithelial morphology. Expression of OCT4 was lost in iPSC cultured witheither FH-CM or combination of 7 CP (FIG. 10B). Differentiated cellscontained albumin as shown by immunostaining, whereas undifferentiatediPSC were negative for albumin staining (FIG. 10C). Moreover, similar todifferentiation of hESC under these conditions, we found differentiatedcells expressed vimentin. These iPSC-derived hepatocytes synthesizedurea and metabolized ethoxyresorufin to resorufin (FIG. 10E, 10F). Thissubstantiated that this combination of 7 CP generated hepatocytes fromiPSC.

Materials and Methods

Chemicals and reagents: CP were purchased and stock solutions wereprepared in either water or ethanol as recommended by the manufacturers(Sigma Chemical Co.; Santa Cruz Biotechnology).

Cells and cell culture: WA-01 hESC were passaged on matrigel-coateddishes in DMEM/F12 medium, 1% B27 supplement, 1% N₂ supplement, 2 mML-glutamine, 0.1 mM NEAA (Life Technologies) and 50 ng/ml basic FGF (R&DSystems). The iPSC were generated from normal human fibroblasts withCytoTune®-iPS Sendai Reprogramming Kit (Life Technologies, Cat #A1378001). The iPSC were cultured on matrigel-coated dishes in DMEM/F12medium, 1% B27 supplement, 1% N₂ supplement, 2 mM L-glutamine, 0.1 mMNEAA (Life Technologies) and 50 ng/ml basic FGF (R&D Systems). To obtainFH-CM, hTERT-FH-B cells were cultured for 24 h in DMEM/F12 medium with2% Knock-out Serum Replacer (KSR), 2 mM L-glutamine, 0.1 mM MEM NonEssential Amino Acids (NEAA), 1% penicillin-streptomycin (LifeTechnologies).

Hepatic differentiation: For differentiation, hESC/iPSC were washed withDMEM/F12 and cultured for 2 weeks in FH-CM and CP in DMEM/F12 with 2%Knock-out Serum Replacer (KSR), 1% B27 supplement, 2 mM L-glutamine, 0.1mM MEM Non-Essential Amino Acids (NEAA), 1% penicillin-streptomycin(Life Technologies). For testing effects of CP in differentiation ofhESC/iPSC, identical culture medium was used except that one or more CPwere added and the step of incubating hTERT-FH-B cells in this mediumwas omitted. HepG2 cells of human origin were included in some studiesand were cultured in usual conditions with serum-containing medium asdescribed in the parent document.

Immunostaining: Cells were fixed in 4% paraformaldehyde in phosphatebuffered saline, pH 7.4 (PBS), blocked/permeabilized with 5% goat serum,0.2% Triton X-100 (Sigma) in PBS for 1 h, and incubated overnight at 4°C. with mouse anti-human albumin antibody (HSA-11 clone, 1:200, Sigma),anti-human OCT3/4 (1:200, Santa Cruz Biotechnology), anti-human vimentin(1:100, US Biologicals). After washes in PBS, TRITC-conjugated goatanti-mouse IgG (1:50, Sigma) cells were counterstained for 1 h with4′-6-diamidino-2-phenylindole (DAPI) (Life Technologies). In negativecontrols, primary antibody was omitted.

Hepatic functions: For ureagenesis, cells were incubated with 5 mMammonium chloride for 12 h and analyzed as described (Cho et al., 2004,(31)). For CYP450 activity, cells were analyzed for 7-ethoxyresorufinconversion, as described (Gupta et al., 1999, (32)).

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What is claimed:
 1. A composition comprising: L-cysteinglutathionedisulfide, γ-Glu-Cys, DL-kynurenine, D-penicillamine disulfide,phenacetin, phytosphingosine HCl, pyridoxal HCl, optionally, anadditional antibiotic.
 2. The composition of claim 1, wherein theadditional antibiotic is tetracaine HCl.
 3. The composition of claim 1,which does not contain the additional antibiotic.